Meiotic maturation in the mammalian oocyte involves a complex series of metabolic and structural changes which ultimately confers fertilizability and development competence to the mature ovum. Since errors in the maturation process result in infertility or defective embryonic development, this proposal seeks to define both the normal and abnormal structural events which occur during the in vitro maturation of mammalian oocytes in order to assess the developmental competence of the oocyte. Three specific aims are proposed: 1. To continue to develop and apply non-invasive methods for the assessment of the developmental capability of in vitro matured mammalian oocytes. Vital fluorescent dyes and digital imaging microscopy will be used to analyze nuclear and cytoplasmic components within living mammalian oocytes during meiotic maturation, in vitro fertilization, and early embryonic cleavages. 2. To analyze chromosome behavior meiosis-1 and meiosis-2 in mammalian oocytes. Using morphological, micromanipulation and digital imaging microscopic techniques, meiotic chromosomes and their relationship to meiotic spindle microtubules will be studied in immature, mature and aged oocytes. 3. To study the physiological regulation of meiotic maturation in cultured mammalian oocytes. Attention will be focused upon both germ cell (oocyte) and somatic cell (cumulus) factors which mediate the complete maturation process emphasizing the areas of intercellular communication, intracellular pH and calcium, and asymmetric changes in the oocyte cortex. Immunofluorescence and electron microscopic methods will used in conjunction with micromanipulation and on hand quantitative fluorescence techniques. These studies will provide new methods for analyzing the developmental competence of oocytes used for in vitro fertilization and embryo transfer in the human and domestic animal species as well as provide a better understanding of the cellular events which lead to chromosome malsegregation during meiosis in the female.